AP1358A - 1-Aryl-2-Hydroxy-3-Isoquinoline Carboxamide HIV Protease Inhibitors, Their Preparation and Use. - Google Patents

Abstract

HIV protease inhibitors, obtainable by chemical synthesis, inhibit or block the biological activity of the HIV protease enzyme, causing the replication of the HIV virus to terminate. These compouns, as well as pharmaceutical compositions that contain these compounds and optionally other anti-viral agents as active ingredients, are suitable for treating patients or hosts with HIV virus, which is known to cause AIDS.

Description

ΑΡ δ Ο 13 5 8 -1-

HIV PROTEASE INHIBITORS

BACKGROUND AND SUMMARY OF THE INVENTION

This invention relates to a novel series of chemicalcompounds useful as HIV protease inhibitors and to the useof such compounds as antiviral agents.

Acquired Immune Deficiency Syndrome (AIDS) is a rela-tively newly recognized disease or condition. AIDS causes agradual breakdown of the body's immune system as well asprogressive deterioration of the central and peripheralnervous systems. Since its initial recognition in the early1980's, AIDS has spread rapidly and has now reached epidemicproportions within a relatively limited segment of thepopulation. Intensive research has led to the discovery ofthe responsible agent, human T-lymphotropic retrovirus III(HTLV-III), now more commonly referred to as the humanimmunodeficiency virus or HIV. HIV is a member of the class of viruses known asretroviruses. The retroviral genome is composed of RNAwhich is converted to DNA by reverse transcription. Thisretroviral DNA is then stably integrated into a host cell'schromosome and, employing the replicative processes of thehost cells, produces new retroviral particles and advancesthe infection to other cells. HIV appears to have aparticular affinity for the human T-4 lymphocyte cell whichplays a vital role in the body's immune system. HIVinfection of these white blood cells depletes this whitecell population. Eventually, the immune system is renderedinoperative and ineffective against various opportunisticdiseases such as, among others, pneumocystic carinipneumonia, Kaposi's sarcoma, and cancer of the lymph system.

Although the exact mechanism of the formation and work-ing of the HIV virus is noc understood, identification ofthe virus has led to some progress in controlling thedisease. For example, the drug azidothymidine (AZT) hasbeen found effective for inhibiting the reverse transcription of the retroviral genome of the HIV virus, AP/PZ 9 9 / 0 1 6 48 SUBSTITUTE SHEET (RULE 25) -2- thus giving a measure of control, though not a cure, forpatients afflicted with AIDS. The search continues fordrugs that can cure or at least provide an improved measureof control of the deadly HIV virus.

Retroviral replication routinely features post-translational processing of polyproteins. This processingis accomplished by virally encoded HIV protease enzyme.

This yields mature polypeptides that will subsequently aidin the formation and function of infectious virus. If thismolecular processing is stifled, then the normal productionof HIV is terminated. Therefore, inhibitors of HIV proteasemay function as anti-HIV viral agents. HIV protease is one of the translated products from theHIV structural protein pol gene. This retroviral proteasespecifically cleaves other structural polypeptides atdiscrete sites to release these newly activated structuralproteins and enzymes, thereby rendering the virionreplication-competent. As such, inhibition of the HIVprotease by potent compounds may prevent proviral integration of infected T-lymphocytes during the early phaseof the HIV-1 life cycle, as well as inhibit viralproteolytic processing during its late stage. Additionally,the protease inhibitors may have the advantages of beingmore readily available, longer lived in virus, and lesstoxic than currently available drugs, possibly due to theirspecificity for the retroviral protease.

In accordance with this invention, there is provided anovel class of chemical compounds that can inhibit and/orblock the activity of the HIV protease, which halts theproliferation of HIV virus, pharmaceutical compositionscontaining these compounds, and the use of the compounds asinhibitors of the HIV protease.

The present invention relates to compounds fallingwithin formula (9) below, and pharmaceutically acceptablesalts, prodrugs, and solvates thereof, that inhibit theprotease encoded by human immunodeficiency virus (HIV) type1 (HIV-1) or type 2 (HIV-2). These compounds are useful inthe treatment of infection by HIV and the treatment of the AP/P/ 9 9 / 0 1 6 48 SUBSTITUTE SHEET (RULE 26) APvΟ 13 5 8 -3- scquired immune deficiency syndrome (AIDS) . The - ounc.u;, their pharmaceutically acceptable salts, and thepharmaceutical compositions of the present invention can beused alone or in combination with other antivirals,imniunomodulators, antibiotics or vaccines, Compounc.s of thepresent invention can also be used as prodrugs. .,ds of treating AIDS, methods of treating HIV infection and methodsof inhibiting HIV protease are disclosed.

The compounds of the present invention are of oneformula (9):

who-re;,, n; R and R! are independently selected from H, nsubstituted or unsubstituted alkyl-ORi group, a coo;ioalkylgroup substituted v . a (Ct-C6)alkyl group or a <1 -Cgalkyl-OH group, a heterocycle group substituted chin aoh-C{j alky 1 group or a (C--C6) alkyl-OH group, an alkyl.-NR-.R-.group, or an alkyl-S (Xl (Y)R4 group, wherein

Rj is H, a substituted or unsubstituted alkyl group, oran acyl group; R, and R3 are each independently selected from H,substituted or unsubstituted alkyl, cycloalkyl ,heterocycle, and aryl groups, and acyl and s · of R< as H, a substituted or unsubstituted alkyl,cycloalkyl, hecerocycle, or aryl group; andX and Y are each independently selected from ~1 and nothing; or a pharmaceutically acceptable prodrug, salt or scutate t. hereof .

8*91-0x66 /d/dV SUBSTITUTE SHEET (RULE 26) ΑΡδ 0 1358 -4-

Preferably in the compounds of formula 9, R is H. Morepreferably, R is H and R.' is a cycloalkyl group selectedfrom:

CHjOH CH3 CH2OH . CHzOH

Preferably in the compounds of formula 9 when at least oneof R and R' is an alkyl-OR! group, R3 is H. Particularlywhen at least one of R and R' is an alkyl-ORX group, thealkyl-ORj is selected from -C (CK3) 2CH2OH, -CH (CH3) CH2OH,-CH2CH2OH, -C(CH3) (CH2OH)2z -C (CH3) 2-O-CH2-O-CH3, -c (ch3) 2ch2-o-CH2-O-CH3, and -C (CH3) 2CH2-O-acyl, or a pharmaceuticallyacceptable prodrug, salt or solvate thereof.

Preferably when at least one of R and R' is acycloalkyl group substituted with a (Cj-CJ alkyl group or a(Cj-C6)alkyl-OH group, the cycloalkyl group is selected from:

Preferably when at least one of R and R' is a heterocycle group substituted with a (Ci~C6) alkyl group or a (C3- C6) alkyl-OH group, the heterocycle group is selected from: APZP/ 9 9 / 0 1 6 48

wherein R3 is H, a substituted or unsubstituted alkyl,cycloalkyl, heterocycle, or aryl group, or an acyl orsulfonyl group. A preferred species of the formula (9) is [3S-[2 (2S*,3S*),3 alpha,4a beta,8a beta]]-N-(l,l-dimethyl-2-hydroxyethy1) decahydro-2-[2-hydroxy-3-[(3-hydroxy-2- SUBSTITUTE SHEET (RULE 26) ΑΡ ΰ 0 1358 -5- methylbenzoyl)amino] -4-(phenylthio)butyl] -3- i s oqu i nol i ne c a rboxand. cte

and its pharmaceutically acceptable salts, and its nrodruganalogs. Preferred prodrugs can be obtained by replacingthe hydrogen in one of the alcohol groups with an acylgroup, and more preferably an amino acid acyl group..

The present invention further provides pharmaceuticalformulations comprising an effective amount of a compound offormula (S) or a pharmaceutically acceptable salt thereof,in combination with &amp; pharmaceutically acceptable carrier,such as a diluent or excipient.

The present invention further provides a method oftreating AIDS comprising administering to a host or patient,such as a primate, an effective amount of a compound of thepresent, invention.

The present invention further provides a method ofinhibiting HIV replication comprising administering to anHIV infected cell, a cell susceptible to HIV infection or ahost or patient, such, as a primate, an effective amount of acompound of the present invention.

Detailed Description of the Invention

The present invention provides new compounds fallingwaachixa formula (9) , as described above, that are useful fortreating HIV infection and/or AIDS.

Applicants incorporate by reference 13.S . Patent No.5,454,926, U.S. Patent Application Nos. 08/708,411 ana08/708.S07, and Japanese Patent Application Nos. JP an.248183 and JP 95-248184, with the caveat that thedefinitions of preferences, terms, variables, labels said the APZP/ 9 9 / 0 1 6 48 SUBSTITUTE SHEET (RULE 26) ΑΡδΟ135 8 -6- like used in each application are applicable only to thecorresponding disclosure from that application.

In particular, since each of the above-identifiedapplications incorporated by reference was preparedseparately, the original applications may use in someinstances the same term, label or variable to mean somethingdifferent. For example, the variable "X" is used in eachapplication, but each application has its own distinctdefinition of the substituent or moiety represented by thisvariable. It will be apparent to those skilled in the artthat the terms, labels and variables in each applicationincorporated by reference are limited solely to thedisclosure from that application, and may be replaced byother suitable terms, labels and variables or the likerepresenting the particular substituents and moieties. Ofcourse, those skilled in the art will realize that anysuitable set of terms, labels and variables may be used togenerically or more specifically represent the subjectmatter disclosed in the present application, includingterms, labels, variables, and the like universallyapplicable to the incorporated disclosures of theabove-identified applications and the following disclosure.

Compounds of the formula (9) may be prodrugs, which canserve to improve the pharmaceutical properties of thecompounds, such as pharmacokinetic properties, for example,improved bioavailability or solubility. .The preparation ofprodrugs may be carried out by standard methods known tothose skilled in the art. A preferred prodrug can beobtained by acylation or alkylation of the starting alcoholwhen R or R1 is CH {CH3) 2CH2OH.

All temperatures stated herein are in degrees Celsius(°C). All units of measurement employed herein are inweight units except for liquids which are in volume units.

The term "alkyl" as used herein refers to straight orbranched chain groups, preferably, having one to eight, morepreferably having one to six, and most preferably havingfrom one to four carbon atoms. The term "Cx-Cj alkyl"represents a straight or branched alkyl chain having from . AP/PZ 9 9 z 0 1 6 48 SUBSTITUTE SHEET {RULE 26) ΑΡ ϋ Ο 1 3 5 8 one to six carbon atoms Exemplary Cp-Cl alkyl groupsinclude reethyl, ethyl, n- propyl, isopropyl, butyl, .isobutyl, sec-butyl, t-butyl, pentyl, neo-pentvl, hexyl,isohexyl, and the like. The term "Cj-CO alkyl" includeswithin its definition the term "C.-C4 alkyl".

The term "cycloalkyl" represents a saturated oxpartially saturated, mono- or poly-carbocylic ring,preferably having 5-14 ring carbon atoms. Exemplarycycloalkyls include monocyclic rings having from 3-7,preferably 3-6, carbon atoms, such as cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and Che " i like. An exemplary cycloalkvl is a C5-C7 cycloalkyl, which is a saturated hydrocarbon ring structure containing fromfive to seven carbon atoms.

The term "alkoxyl" represents -0-alkyl. An example ofan alkoxyl is a Cj-Ct; alkoxyl, which represents a straight orbranched alkyl chain having from one to six carbon atomsattached to an oxygen atom. Exemplary Ci-Ce alkoxyl -groupsinclude methoxyl, ethoxvl, propoxy1, isopropoxyl, .oxyl,sec-butoxyl, t-butoxyl, pentoxyl, hexoxvl, and the Like. C:!-Cf; alkoxyl includes within its definition a Cj-Ci aiKoxyl.

The term "aryl" as used herein refers to a carbocyclicor heterocyclic, aromatic, 5-14 membered monocyclic orpolycyclic ring. Exemplary aryls include phenyl, naphthyl, ; ') anthryl, phenanthryl, thienyl, pyrrolyl, imidazolyl. pyrazoiyl, furyl, isothiazolyl, furazanyl, isoxazoltl,thiazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl,triazinyl, benzo[b]thienyl, naphtho[2,3-b]thianthrenyi,isobenzofuranyl, chromenyl, xanthenyl, phenoxathierindolizinyl, isoindolyl, indolyl, indazolyl, purinyl,iscquinolyl, quinolyl, phthalazinyl, naphthyridinyl.quincxyalinyl, quinzolinyl, benzothiazolyl, benzinu.· tlyl,cetrahydroquinolinyl, cinnolinyl, pteridinyl, carbarolyl,beta-carbolinyl, phenanthridinyl, acridinyl, perimidtnyl,phenanthrolinyl, phenazinyl, isothiazolyl, phenottu ntl,and. phenoxazinyi .

The term "aryloxyl" represents -0-aryl. AP/PZ 99/01648 SUBSTITUTE SHEET (RULE 26) AP 0 0 1 3 5 8 WO ·'? ,) -8-

The term "hydrolyzable group" is a group, which whenbonded to an oxygen, forms an ester, which can be hydrolyzedin vivo to a hydroxyl group. Exemplary hydrolyzable groups,which are optionally substituted, include acyl function,sulfonate function and phosphate function. For example,such hydrolyzable groups include blocked or unblocked aminoacid residue, a hemisuccinate residue, and a nicotinateresidue.

The term "halogen" represents chlorine, fluorine,bromine or iodine. The term "halo" represents chloro,fluoro, bromo or iodo.

The term "carbocycle" represents an aromatic or asaturated or a partially saturated 5-14 membered monocyclicor polycyclic ring, such as a 5- to 7-membered monocyclic or7- to 10-membered bicyclic ring, wherein all the ringmembers are carbon atoms.

The term "heterocycle" represents an aromatic or asaturated or a partially saturated, 5-14 membered, monocylicor polycyclic ring, such as a 5- to 7-membered monocyclic or7- to 10-membered bicyclic ring, having from one to threeheteroatoms selected from nitrogen, oxygen and sulfur, andwherein any nitrogen and sulfur heteroatoms may optionallybe oxidized, and any nitrogen heteroatom may optionally beguaternized. The heterocyclic ring may be attached at anysuitable heteroatom or carbon atom. Examples of suchheterocycles include decahydroisoquinolinyl, octahydro-thieno[3,2-c]pyridinyl, piperidinyl, piperazinyl,azepinyl, pyrrolyl, pyrrolidinyl, pyrazolyl, pyrazolidinyl,imidazolyl, isobenzofuranyl, furazanyl, imidazolinyl,imidazolidinyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, oxazolyl, oxazolidinyl, isoxazolyl, thianthrenyl, triazinyl, isoxazolidinyl, morpholinyl,thiazolyl, thiazolidinyl, isothiazolyl, quinuclidinyl, iso-thiazolidinyl, indolyl, quinolinyl, chromenyl, xanthenyl,isoquinolinyl, benzimidazolyl, thiadiazolyl, benzopyranyl,benzothiazolyl, benzoazolyl, furyl, tetrahydrofuryl,tetrahydropyranyl, thienyl, benzothienyl, benzo [b]thienyl,naphtho[2,3-b]thienyl, thiamorpholinyl, APZP/ 9 9 / 0 1 6 48 SUBSTITUTE SHEET (RULE 2B) ΑΡ δ 0 13 5 8 „q_ tniamorpholinylsulfoxide, thiamorpholinylsulfone, oxadiazolyl, triazoiyl, tetrahydroquinolinyl, tetrahydriso-qumoiinyl, phenoxathienyl, indolizinyl, isoindolylindazolyl, purinyl, isoquinolyl, quinolyl, phthalazinyl,naphthyridinyl, quinoxyaiinyl, quinzolinyl,tetrahydroquinolinyl, cinnolinyl, pteridinyl, carbazclyl,beta-carbolinyl, phenanthridinyl, acridinyl, perimidinyl,phenanthrolinyl, phenazinyl, isothiazolyl, phenothiszi.nyl,α,τΑ phenoxazinyl.

The term "thioether" includes S-aryl, such asphenylrhic and naphthylthio; S-heterocycle where c,neheterocycle is saturated or partially saturated; S-(C5-C7)--cycloalkyl; and S-alkyl, such as Cj-CEl alkyl thio.

In the thioether, the -aryl, the -heterocycle, the-cycloalkyl and the -alkyl can optionally be substituted.

An example of a thioether is "C;-C6 alkylthio", whichrepresents a straight or branched alkyl chain having fromone to six carbon atoms attached to a sulfur atom.

Exemplary C,-C6 alkylthio groups include methylthic,ethylthio, propylthio, isopropylthio, butylthio, sec -bucylthio, t-butylthio, pentylthio, hexylthio, and the like.

The term "mercapto" represents -SH.

The term "amino" represents -NLjLj, wherein L, and L,axe preferably independently selected from oxygen,carbocycle, heterocycle, alkyl, sulfonyl and hydrogen,· orNC (0)1,3, wherein L, is preferably alkyl, alkoxyl, hydrogen or-K'L-.V,. The aryl, alkyl and alkoxyl groups can optionally besubstituted. An example of an amino is C,-C4 alkylamino,which represents a straight or branched alkyl chair; havingfrom one to four carbon atoms attached to an amino group.Exemplary C;-C4 alkylamino groups include methylamino,ethylamino, propylamine, isopropylamino, butylamino, sec-butylamino, and the like. Another example of an ammo isdi(C,-C,)alkylamino, which represents two straight orbr&amp;nched alkyl chains, each having from one to four carbonatoms attached to a common amino group. Exemplary di(C, -Cf) alkylamino groups include dimethylamino,ethylmethylamino, methylpropylamino, ethylisopropvlsmino, AP/P/ 9 9 / 0 1 6 48 SUBSTITUTE SHEET (RULE 26) ΑΡ Ο Ο 13 5 8

-10- butylmethylamino, sec-butylethylamino, and the like. Anexample of an amino is C,-C4 alkylsulfonylamino, which has astraight or branched alkyl chain having from one to fourcarbon atoms attached to a sulfonylamino moiety. ExemplaryC1-C4 alkylsulfonylamino groups include methylsulfonylamino,ethylsulfonylamino, propylsulfonylamino, isopropylsulfonylamino, butylsulfonylamino, sec- butylsulfonylamino, t-butylsulfonylamino, and the like.

The term "acyl" represents L6C(O)L4, wherein L6 is a single bond, -0 or -N, and further wherein L4 is preferablyalkyl, amino, hydroxyl, alkoxyl or hydrogen. The alkyl andalkoxyl groups can optionally be substituted. An exemplaryacyl is a Cj-C4 alkoxycarbonyl, which is a straight orbranched alkoxyl chain having from one to four carbon atomsattached to a carbonyl moiety. Exemplary C3-C4 alkoxycarbonyl groups include methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl, and the like. Another exemplary acyl is acarboxy wherein L6 is a single bond and L4 is alkoxyl,hydrogen, or hydroxyl. A further exemplary acyl is N-(C3-C4)alkylcarbamoyl (Ls is a single bond and L„ is an amino),which is a straight or branched alkyl chain having from oneto four carbon atoms attached to the nitrogen atom of acarbamoyl moiety. Exemplary N-(C3-C4) alkylcarbamoyl groupsinclude N-methylcarbamoyl, N-ethylcarbamoyl, N-propylcarbamoyl, N-isopropylcarbamoyl, N-butylcarbamoyl,and N-t-butylcarbamoyl, and the like. Yet another exemplary-acyl is N, N-di(C!-C4)alkylcarbamoyl, which has two straightor branched alkyl chains, each having from one to fourcarbon atoms attached to the nitrogen atom of a carbamoylmoiety. Exemplary N,N-di (C3-C4) alkylcarbamoyl groups includeN,N-dimethylcarbamoyl, N,N-ethylmethylcarbamoyl, N,N-methylpropylcarbamoyl, N,N-ethylisopropylcarbamoyl, N,N-butylmethylcarbamoyl, N,N-sec-butylethylcarbamoyl, and thelike.

The term "sulfinyl" represents -SO-L5, wherein L5 ispreferably alkyl, amino, aryl, cycloalkyl or heterocycle.

8» 9 I 0 / 6 6 /d/dV SUBSTITUTE SHEET {RULE 26)

ΑΡ ϋ Ο 13 5 S -11-

The alkyl, aryl, cycloalkyl and heterocycle can alloptionally be substituted.

The term "sulfonyl" represents -SO2-L5, where ir: G, ispreferably alkyl, aryl, cycloalkyl, heterocycle ox amino.

The alkyl, aryl, cycloalkyl and heterocycle can alloptionally be substituted. An example of a sulfonyl is aC-,~C- alkylsulfonyl, which is a straight or branched alkylchain having from one to four carbon atoms attached tc asulfonyl moiety. Exemplary Cj-C4 alkylsulfonyl groupsinclude methylsulfonyl, ethylsulfonyl, propylsulfonyl,isopropylsulfonyl, butylsulfonyl, sec-butylsulfonyl, t- } butylsulfonyl and the like.

As indicated above, many of the groups are optionally substituted. In fact, , unless specifically noted, ail of thegroups defined by the terms defined in this application maybe substituted or unsubstituted. For instance, when theterm "alkyl" is used, it should be understood to encompassboth substituted and unsubstituted alkyl unless specificexclusion of one or the other is positively stated.

Examples of substituents for alkyl and aryl includemercapto, thioether, nitro (N02) , amino, aryloxyl, halogen,hydroxyl, alkoxyl, and acyl, as well as aryl, cycloalkyl andsaturated and partially saturated heterocycles. Examples ofsubstituents for heterocycle and cycloalkyl include thoselisted above for alkyl and aryl, as well as aryl and alkyl. , Exemplary substituted aryls include a phenyl or

. I naphthyl ring substituted with one or more substituents,preferably one to three substituents, independently selectedfrom halo, hydroxy, morpholino(C^C^alkoxy carbonyl, pyridyl(G-Cj alkoxycarbonyl·, halo (Cj-Cy) alkyl, Cj-C,, alkyl, G ~Cfalkoxy, carboxy, C,-C4 alkoxycarbonyl, carbamoyl, N-(G-CJ alkylcarbamoyi, amino, Cj-C4 alkylamino,di (C.-C.) alkylamino or a group of the formula where a is 1, 2, 3 or 4; and R7 is hydroxy, Cj-C4alkoxy, carboxy, Ci-C4 alkoxycarbonyl, amino, carbamoyl, G-C, alkylamino or di. (C,-C4) alkylamino .

Another substituted alkyl is halo(Cj-CJ alkyl, whichrepresents a straight, or branched alkyl chain having from AP/P/ 9 9 / 0 1 6 A 8 SUBSTITUTE SHEET (RULE 26) ΑΡ Ο 0 13 5 8 -12- one to four carbon atoms with 1-3 halogen atoms attached toit. Exemplary halo(Cj-Cj alkyl groups include chloromethyl,2- bromoethyl, 1-chloroisopropyl, 3-fluoropropyl, 2,3-dibromobutyl, 3-chloroisobutyl, iodo-1-butyl,trifluoromethyl and the like.

Another substituted alkyl is hydroxy (Cj-C^) alkyl, whichrepresents a straight or branched alkyl chain having fromone to four carbon atoms with a hydroxy group attached toit. Exemplary hydroxy(Cj-CJ alkyl groups include hydroxymethyl, 2-hydroxyethy1, 3-hydroxypropyl, 2-hydroxy-isopropyl, 4-hydroxybutyl and the like. " j Yet another substituted alkyl is Cj-C4 alkylthio(C;- CJ alkyl, which is a straight or branched Ci~C4 alkyl groupwith a Cj-Ci alkylthio group attached to it. Exemplary Cx-C^alkylthio(C,-C4) alkyl groups include methylthiomethyl,ethylthiomethyl, propylthiopropyl, sec-butylthiomethyl, andthe like.

Yet another exemplary substituted alkyl isheterocycle (C^- C4) alkyl, which is a straight or branchedalkyl chain having from one to four carbon atoms with aheterocycle attached to it. Exemplary heterocycle (Cj-C,,) alkyls include pyrrolylmethyl, quinolinyl-methyl, 1-indolylethyl, 2-furylethyl, 3-thien-2-ylpropyl, 1-imidazolylisopropyl, 4-thiazolylbutyl and the like. . Yet another substituted alkyl is aryl(Cj-CJ alkyl, whichis a straight or branched alkyl chain having from one tofour carbon atoms with an aryl group attached to it.Exemplary aryl (Cj-C4) alkyl groups include phenylmethyl, 2-phenylethy1, 3-naphthyl-propyl, 1-naphthylisopropyl,4-phenylbutyl and the like.

The heterocycle can, for example, be substituted with1, 2 or 3 substituents independently selected from halo,halo (Ci- C4) alkyl, Cj-C, alkyl, Cj-C^ alkoxy, carboxy, C,-C„alkoxycarbonyl, carbamoyl, N-(Cj-C4)alkylcarbamoyl, amino,C^-C^alkylamino, di(Cj-C4)alkylamino or a group having thestructure -(CH2)a-R7 where a is 1, 2, 3 or 4 and R7 ishydroxy, Cj-Ci alkoxy, carboxy, Cl-C4 alkoxycarbonyl, amino,carbamoyl, Cx-C4 alkylamino or di(Cj-C4)alkylamino. AP/P/9 9 / 0 1 6 48 SUBSTITUTE SHEET (RULE 26) -13~

Examples of substituted heterocycles include3-N-t-butyl carboxamide decahydroisoquinolinyl, 6-K-t-butylcarboxamide octahydro-thieno[3,2-c]pyridinyl, 3- methylimidazolyl, 3-methoxypyridyl, 4-chloroquinoiinvl, 4- aminothiazolyl, 8-rnethylquinolinyl, 6-chloroquinoxalinyl, 3- ethylpyridyl, 6-methoxybenzimidazolyl, 4-hydroxyfuryl, 4- methylisoquinolinyi, 6,8-dibromoquinolinyl, 2-methy'i-- 1,2,3,4-tetrahydroisoquinolinyl, N-methyl-quinolin-2.....yl, 2- t-butoxycarbonyl-1,2,3,4-isoquinolin-7-yl and the like.

Exemplary heterocyclic ring systems represented by A orB include (1) 5-membered monocyclic ring groups sues asthienyl, pyrrolyl, imidazolyl, pyrazolyl, furyl,isothiazolyl, furazanyl, isoxazolyl, thiazolyl and the like;(2> 6-membered monocyclic groups such as pyridyl, pyrazinyl,pyrimidinyl, pyridazinly, triazinyl and the like; and (3)polycyclic heterocyclic rings groups, such as decahydroisoquinolinyl, octahydro-thieno [3,2-c] pyridinyl,benzo[h]thienyl, naphtho [2,3-b]thianthrenyl,isobenzofuranyl, chromenyl, xanthenyl, and fully orpartially saturated analogs thereof. A cycloalkyl may be optionally substituted with 1, 2 or3 substituents independently selected from halo,halo (C;-C,) alkyl, Ct-C,. alkyl, C^-C,. alkoxy, carboxy, C,~C4alkoxycarbonyl, carbamoyl, K-(Cj-C4)alkylcarbamoyl, amino,C_-C4 alkylamino, di (Ci~C4) alkylamino or a group having thestructure - (CH2) a-R'where a is 1, 2, 3 or 4 and R' ishydroxy, Crt-C4 alkoxy, carboxy, C:-C4 alkoxycarbonyl, amino,carbamoyl, Cy-C4 alkylamino or di (C,-C4) alkylamino.

Exemplary substituted cycloalkyl groups include 3- methylcyclopentyl, 4-ethoxycyclohexyl, 5- carboxycyclo-heptyl, 6-chlorocyclohexyl and the like.

Exemplary substituted hydrolyzable groups includeK-benzyl glycyl, N-Cbz-L-valyl, and N-methyl nicotinate.

The compounds of the present invention have at leastfive asymmetric centers denoted by an asterisk in theformula (9) below:

8*910/66 /d/dV SUBSTITUTE SHEET (RULE 26) APO 0 1358

As a consequence of these asymmetric centers, thecompounds of the present invention can occur in any of thepossible stereoisomeric forms, and can be used in mixturesof stereoisomers, which can be optically active or racemic,or can be used alone as essentially pure stereisomers, i.e., '' , at least 95% pure. All asymmetric forms, individual stereoisomers and combinations thereof, are within the scopeof the present invention.

The individual stereoisomers may be prepared from theirrespective precursors by the procedures described above, byresolving the racemic mixtures, or by separating thediastereomers. The resolution can be carried out in thepresence of a resolving agent, by chromatography or byrepeated crystallization or by some combination of thesetechniques which are known in the art. Further detailsregarding resolutions can be found in Jacques et al.,Enantiomers, Racemates, and Resolutions, John Wiley &amp; Sons1981. 5 Preferably, the compounds of the present invention are substantially pure, i.e, over 50% pure. More preferably, the compounds are at least 75% pure. Even more preferably, the compounds are more than 90% pure. Even more preferably,the compounds are at least 95% pure, more preferably, atleast 97% pure, and most preferably at least 99% pure.

As mentioned above, the invention includes thepharmaceutically acceptable salts of the compounds definedby formula (9). A compound of this invention may possess asufficiently acidic, a sufficiently basic, or bothfunctional groups, and accordingly react with any of anumber of inorganic or organic bases, and inorganic andorganic acids, to form a pharmaceutically acceptable salt. AP/P/9 9 /0 16 48 SUBSTITUTE SHEET (RULE 26) ΑΡϋ Ο 135 8

The term "pharmaceutically acceptable salt", a;; usedherein, refers to salts of the compounds of the aboveformula which are substantially non-toxic to livingorganisms. Exemplary pharmaceutically acceptable saltsinclude those salts prepared by reaction of the compounds ofthe present invention with a mineral or organic acid or aninorganic base. The reactants are generally combined in amutual solvent such as diethylether or benzene, for acidaddition salts, or water or alcohols for base additionsalts. The salts normally precipitate out of solutionwithin about one hour to about ten days and can be isolated ·-'·, by filtration or other conventional methods. Such salts are known as acid addition and base addition salts.

Acids that may be employed to form acid addition saltsare inorganic acids such as hydrochloric acid, hydrcbromicacid, hydroiodic acid, sulfuric acid, phosphoric acid, andthe like, and organic acids such as p-toluenesulfonic,methanesulronic acid, oxalic acid, p-brotnophenylsul tonicacid, carbonic acid, succinic acid, citric acid, benzoicacid, acetic acid, and the like.

Examples of pharmaceutically acceptable salts are thesulfate, pyrosulfate, fcisulfate, sulfite, bisulfite,phosphate, monohydrogenphosphate, dihydrogenphosphate,metaphosphate, pyrophosphate, chloride, bromide, iodide, Ί acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caproate, heptanoate, propiolate, ,.,7 oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate, hexyne-1,6-dioate, benzoate,chlorobenzoate, methylbenzoate, dinitrobenzoate,hydroxybenzoate, methoxybenzoate, phthalate, sulfonate,xylenesulfonate, phenylacetate, phenylpropionate,phenylbur.vrate, citrate, lactate, g-hydroxybutyrate,glycollate, tartrate, methane-sulfonate, propanesulfonate,naphthalene-1-sulfonate, napththalene-2-sulfonate, mandelateand the like.

Preferred pharmaceutically acceptable acid additionsalts are those formed with mineral acids such ashydrochloric acid ana hydrobromic acid, and those farmed AP/P/ 99/01648 SUBSTITUTE SHEET (RULE 26) ΑΡ Ο 0 13 5 8 -16- with organic acids such as maleic acid and methanesulfonicacid.

Base addition salts include those derived frominorganic and organic bases, such as ammonium or alkali oralkaline earth metal hydroxides, carbonates, bicarbonates,and the like. Such bases useful in preparing the salts ofthis invention thus include sodium hydroxide, potassiumhydroxide, ammonium hydroxide, potassium carbonate, sodiumcarbonate, sodium bicarbonate, potassium bicarbonate,calcium hydroxide, calcium carbonate and the like. Thepotassium and sodium salt forms are particularly preferred. ·"' j A "pharmaceutically acceptable prodrug" is intended to mean a compound that may be converted under physiologicalconditions or by solvolysis to a compound of the formula 9. A "pharmaceutically acceptable solvate" is intended tomean a solvate that retains the biological effectiveness andproperties of the biologically active components ofcompounds of formula 9.

Examples of pharmaceutically acceptable solvatesinclude, but are not limited to, compounds of formula 9 incombination with water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, or ethanolamine.

It should be recognized that the particular counterionforming a part of any salt of this invention is not of acritical nature, so long as the salt as a whole ispharmacologically acceptable and as long as the counterion 'does not contribute undesired qualities to the salt as awhole. AP/P/ 99/01648 SUBSTITUTE SHEET (RULE 26) APvΟ 1 J 5 8 A preferred compound is compound 21

H

[2S~ [2 i2S* , 3S*) , 3 alpna,4a beta, 8a beta]]-N-{l,l-dimethyi-2-hydroxyethyl)decahydrc-2-[2-hydroxy-3-[(3-hydroxy-2-methvlbensoyl) amino} -4-- (phenylthio)butyl] -3-isoquinolinecarboxamide. A process for making compound 21 is provided below.

Compound 21 has also been obtained as a metabolite from theplasma of patients administered [3S- (3R,4aR«,8aR*,2'S*,3'S*}]-2- [2'-hydroxy-3'-phenylthiomethyl- 4'-aza-5'-oxo-5'-(2''-methyl-3''-hydroxyphenyl)pentyl]decahydroisoquinoline-3-N-t-butylcarboxamide methanesultonicacid salt, which is disclosed in U.S. Patent No. 5.484,926.

The compounds of formula 9 can be prepared according tothe following Reaction Scheme I. 0 AP/P/ 9 9/01848 SUBSTITUTE SHEET (RULE 26)

REACTION SCHEME I

ΑΡϋ01358 -18-

Scheme I. General Synthetic Pathway for the Production of 9b and Derivative CONH-t-Bu aqueoushylrolyss

la

Mnc

Deprctea bnSiep3

Step la

R

4

R

Step 4

Me

Compound la, perhydroisoquinoline, which is commercially available from NSC Technologies (Chicago, IL)or Procos SpA (Milan, Italy) is subjected to prolonged acidhydrolysis in step la to obtain compound 2a. A variety of inorganic acids may be used in either an aqueous/organicsolvent mixture or in water alone at temperatures above 50°C. An example of such an inorganic acid is 6N aqueous HC1.Substitutes for compound la include the corresponding esters

8*910/66 /d/dV lb, thioesters lc or other amides Id: cooz lb

where Z, Zx and Z2 may each independently be alkyl,cycloalkyl, heterocycle, or aryl. SUBSTITUTE SHEET (RULE 26) APO01358 -15-

Compound 2a is then protected at the amine nitrogen toobtain compound 2b in seep 2b. The protecting group Ro is defined as a suitably conjugating group to avoid unwamsddecomposition of activated carboxylate derivatives ofcompound 2b in Step 2, Such protecting groups typically canbe carbamate in origin, having a general structure offormula 11:

11

The identity of R" in formula 11 can be any alkyl, cycloalkyl, aryl, or heterocycle which can be removed easilyin a deprotection step after Step 2. Examples of R" include, but are not limited to methyl, ethyl, propyl,isopropyl, η-butyl, isobutyl, t-butyl or higher branched orunbranched alkyl, 2,2,2-trichloroethyl, 2- trimethylsilylethyl, allyl, phenyl, substituted phenyl,benzyl, substituted benzyl, 9-fluorenylmethyl, 9-anthrylmethyl and higher polycyclic aromatic ring system.

The following materials, as defined below, can be obtainedfrom the Aldrich Chemical Co. (Sigma Aldrich Fluka): σ 2,2,2-tnchloroethyl — CH2—C-0 σ K CH3 2-trin»tbvtsHyi©thyi~ — C- Si—- CH3 H CHa aM" — CHj-CHsCHs

8Ϋ 9 I 0 / 6 6 /d/dV benzyl-· — CHj.

Mtourenylrrethyl 9-anthryinKinyi SUBSTITUTE SHEET (RULE 26) ΑΡ Ο Ο 13 5 8 -20-

Such protecting groups typically can be installed byacylation reaction of the corresponding haloformate ester12a or a dicarbonate 12b: an

O a fto' x12a X = halogen 0

X

R’O^Y 12b Y=OCOR" in the presence of a suitable base in typical organicsolvents for these types of reactions such as halogenatedsolvents, ethers and hydrocarbons. Such bases are typicallyinorganic, such as metal hydroxides, bicarbonates andcarbonates or organic bases such as amines like triethylamine, diethylamine, diethyl isopropylamine, 1,8-diazabicyclo[2.2.2]octane (DABCO) or related di- ortrialkyl-amines, as well as amidine bases like 1,8-diazabicyclo[5.4.0] undec-7-ene (DBU) and 1,8-diazabicyclo[4.3.0)non-5-ene (DBN). The followingmaterials, as defined below, can be obtained from theAldrich Chemical Co. (Sigma Aldrich Fluka):

CABOO = jjX

o CQ AP/P/ 99 / 0 1 6 48

These reactions are typically run anywhere from belowroom temperature to approximately 100 °C.

The amide coupling Step 2 can be accomplished in anynumber of fashions depending on how the carboxyl group isactivated. A group J is installed in Step 2 by reaction ofthe carboxylic acid 2b to produce the activated derivative2c. SUBSTITUTE SHEET (RULE 26) APvΟ 13 5 8 COGii ηΥ activation of fe carboxyl group 2b Bp - ainue protecting group ip J can cq, 2c J = having group R ,R'Rd' !

H base

R

Oy 0 : any of a variety of leaving groupishalogen, pseudohalogen (incited mgi’de, cyanide, iso·,-. Re and isothiocyanate}, alkyl crrenesclfonate, aromatic heterocycle(bonded through aand N-hydroxyheterocycle, including bydroxybenzotriazole ester. Hieapply to the terms above: as a1koxy, hydroxy, QG id hiΓΟ2.Ι cm) . roxy su c c ίnimi de nrl.oviing definitions cyanideis oeyanate isothiocyanate alKvIsulfo.nate

• 1— tvs N - Cit Nyte 0=0

i k·· Cse S

C ii O · S-aikyl 0 AP/P/ 9 9'0 16*8 arennsuftonate - aryl id ··-«·.->□ roxyheterocycffc where N « nitrogen beteronycie ο o N-hyd lOxysuccinimiht (--=0- n hydroxybe nzot ria zo 0

N

OH SUBSTITUTE SHEET (RULE 25) APO 0 13 5 8 -22-

The acyl halides (2c, J = halogen) may be preparedusing inorganic halogenating agents such as thionyl chlorideor bromide, phosphorous trichloride or bromide, phosphorouspentachloride or bromide or organic agents such as oxalylchloride or trichlorisocyanuric acid. Esters (2c, J = OR") (R" is defined above) may be prepared in a variety of waysstarting from the acid chloride 2c where J is Cl by combination with the desired alcohol in the presence of anorganic or inorganic base stated previously for theacylation of compound 12a or compound 12b. Alternatively,the ester may be produced by acid-promoted esterification inthe presence of the desired alcohol. The sulfonates (2c, J =OSO2W1( where Wj is alkyl or aryl) are typically made byreaction of the carboxylic acid 2b with alkyl orarylsulfonyl chlorides in the presence of an organic aminebase such as triethylamine in a non-polar solvent attemperatures below 0 °C. Alkyl and arylsulfonyl are definedas follows: o

altyajlfonylchloride = O—S—alkylO

O arenesulfonyl chloride = Q—S—aryt

II 0 AP/P/ 9 9/01648

The pseudohalogen derivatives of 2c (J = pseudohalogen)are typically made from the acid halides 2c (J = halogen) by reaction with inorganic pseudohalide in the presence of abase. Such bases include, but are not limited to metalhydroxides, bicarbonates and carbonates or organic basessuch as amines like triethylamine, diethylamine, diethylisopropylamine, 1,8-diazabicyclo[2.2.2]octane (DABCO) orrelated di- or trialkylamines, as well as amidine bases like1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) and 1,8-diazabicyclo[4.3.0]non-5-ene (DBN). A particularlypreferred base is triethylamine. The heteroaromatic SUBSTITUTE SHEET (RULE 26)

At- Μ ϋ 1 3 5 8 derivatives cf 2c are also made from the acid halides 2c (J• halogen), utilizing the specific heteroaromatic ·. in.in the presence of an amine base in a non-polar schc/crrt .

The N-hydroxyheterocyclic derivatives of 2c can be th - , the acid halides as above and may also be generate: -. r.galkyl carbodiimides (alkyl-N=C=N-alkyl, where thegroups can be the same or different) or aryl carbo;: oie;(aryi“N=C=N-aryl, where the aryl groups can be the same ordifferent) and an amine base as condensing agents.

The primary or secondary amine (shown above the arrowin Step 2 of Scheme 1) used in the coupling process mayincorporate suitable protecting groups, depending on thefunctionality present in the amine and the mode of coupling-used, Trie mode of coupling of 2c with a primary or secondary amine can be carried out in a variety c; depending cn the identity of J. When a free acid is used(2c, J =- OK) the coupling can be performed using -carbodr imide-based methods utilizing any of the commonreagents of this class, including dicyclohexylcam nidior related dialkylcarbodiimides, EDC (salts of 1-(3-direrhylannopropyl}--3-ethyicarbodiimide) or related water-sc-ruble reagents along with an organic amine base in polarorganic solvents such as dioxane, DMF, NMP and at'-" . - in the presence of an M.....hydroxyheterocyclic compound such as K-iiydroxysuccinimide or 3-hydroxybenzotriazole.

Alternatively, haloformate esters, such as 12d, may Pe usedto temporarily activate the acid to give mixed anhyari-des ofgeneral formula 2d. .0

R'O' 0 12d X = halogen

8*910/66 zd/dV SUBSTITUTE SHEET (RULE 26) ΑΡθ 0 1358 -24-

Such haloformate esters are typically as shown in 12d aboveand include methyl-, ethyl-, isopropyl-, isobutyl-, n-butyl,phenyl- and related alkyl and aryl chloroformates, definedbelow. alkyl chloroformate =

O

X alky 1-0" Ο

O aryl chloroformate = . aryl-0 0

Formula 2d is a possible intermediate in the step fromformula 2b to formula 3. Formula 2d is an intermediate, butthe process described here results in formula 3, withoutisolation of Formula 2d.

These reactions are typically performed in a variety ofnon-polar organic solvents like halocarbons and ethers suchas diethyl ether, methyl t-butylether, diisopropyl ether,dioxane and THF at temperatures below 0 °C accompanied by anorganic amine base such as triethylamine, diethylamine,diethyl isopropylamine, DABCO or related di- or trialkylamines, as well as amidine bases like DBU and DBN.

When J in compound 2c is an alkyl or arenesulfonate (J= OSO2R or 0S02Ar) , the coupling can be performed in avariety of non-polar organic solvents like halocarbons andethers, such as diethyl ether, methyl t-butylether,diisopropyl ether, dioxane and THF at temperatures below 0°C, accompanied by an organic amine base such astriethylamine, diethylamine, diethyl isopropylamine, DABCOor related di- or trialkylamines, as well as amidine baseslike DBU and DBN.

When J in compound 2c is a halogen or pseudohalogen,the coupling may be performed in most common organicsolvents such as THF, diethyl ether, dioxane, methyl t-butylether or other ethers; acetone, cyclohexanone, methylisobutylketone and other ketones; esters such as ethyl, 8*810/66 /d/d tf SUBSTITUTE SHEET (RULE 26)

ΑΡ Ο 0 I 3 5 S -25“ methyl and isoprop/. . .ate; haloger.ated solvents . ashajogenated. methanes and ethanes, chlorobenzene and otherha.lngenated benzenes; nitriles such acetonitrile andprcpicnitrile; lows· hols such as ethanol, isot u.. t-butanol and related alcohols; and polar organic solventssuch as dimethylforrnamicie . dimethylsulfoxide, N-rasthylh-pyrrollidone and related amide-containing solvents, A oas-is frequently used and. may be any of a number of inorganic . , as metal hydroxides, bicarbonates and t- . ites or organic bases such as amines like triethylamine..diethylamine, diethyl isopropylamine, DABCO or related di-or trialkylamines, as well as amidine bases like DBA andDBS .

One skilled in. the art will be able to performamide coupling Step 2 with other possible J groups.

In Step 3 prote group removal can be ace . ·-; using any of the standard methods for deprotecting aparticular class of protecting group. Simple alkyl - anasubstituted alkyl carbamates can be removed with aqueoussolutions of base at. temperatures up to about 100 nC.employing any of the common inorganic metal hydroxides suchas eodutinc·, lithium-, potassium- or barium hydroxide or , ' .1-.. f other metals in at least stoichiometric amounts. Carbamate protecting groups that contain benzylgroups bonded to oxygon may be removed by hydrogen:' .. .with a palladium or platinum catalyst. Alternatively,aqueous base hydrolysis may be used at temperatures up toabout 100 -’C, employing any of the common inorganic ~ ohhydroxides such as sodium-, lithium-, potassium- or bariumhydroxide or hydroxides of other metals in at leaststoichiometric amounts. A variety of anhydrous acids- “nayalso be used for deprotection of benzyl-based carbamates,including HCl, HBr and HI. Lewis acids of boron andaluminum such as Aid.. , B3r3, BCr, in non-polar sc . are also effective. Certain substituted benzyl, aryl or. alkylgroups m which the specific substitution pattern is chosenfur its ability to be removed under specific conditions mayalso be used. For example, the 2- AP/P/ 99/01648 SUBSTITUTE SHEET (RULE 26) APO01358 -26- trimethylsilylethylcarbonyl group (Teoc) is a protectinggroup designed to take advantage of the specific reactivityof the 2-trimethyisilylethyl group in the deprotectionprocess. 2-Trimethylsilylethylcarbonyl chloride may be usedto protect the amine nitrogen and may later be removed usingsources of fluoride ion such as HF or tetraalkylammoniumfluoride salts.

In Step 4, the perhydroisoquinoline piece of formula 4is connected to the Chloroalcohol (compound 5, Scheme I) viaan epoxide intermediate (13) generated via the base-inducedclosure of the vicinal chlorohydrin functionality. SPh

CbzHN 13

Compound 5 is produced by Kaneka Industries, Japan. Severalclose-open procedures in proceeding from compound 5 -compound 13 - compound 6 may be used. The epoxide 13 may beisolated or it may be reacted with 4 added either subsequentto formation of 13 or 4 may be present from the beginning ofthe sequence. The epoxide 13 can be generated using inorganic bases such as metal hydroxides, carbonates andbicarbonates in solvents such as alcohols like methanolethanol or isopropyl alcohol, ethers such as THF and dioxaneor mixtures of the two. The epoxide can also be generated ina 2-phase solvent system consisting of water and ahalocarbon solvent such as dichloromethane along with thebase. A phase-transfer catalyst such as a tetraalkylammonium salt may be used to facilitate theprocess. The process of opening the epoxide 13 withcompound 4 is accomplished in alcohol solvents or mixtures of an alcohol and another solvent which may be an ether or adipolar aprotic solvent such as dimethylformamide ordimethylsulfoxide. The opening of the epoxide 13 with AP/P/ 9 9 / 0 1 648 SUBSTITUTE SHEET (RULE 26) AP i-· 0 13 5 8 -27- compound 4 co give compound 6 is optimally performed over aperrod of 2-7 hours ar, 50 - 60 "C.

In Step 5 the carbobenzyloxy group can be removed togive the tree amine 7. This can be done using HBr it acetic acid using cosolvents such as halocarbons. It can ; beperformed using halides of boron such as BBr, and BC'h orallyl substituted boron halides such as dimethylboroubromide in halocarbon solvents like chloroform anddichlorornethane at temperatures ranging from 0 °C up toambient temperature. Alternatively, the carbobenzygroup can be removed by hydrolysis using aqueous/aicoholios. ,.. ·. i metal hydroxides like barium, sodium, lithium or potassium hydroxide at temperatures above ambient forperiods of hours.

Step 6a is the coupling of benzoic acid der.. -formula 8 to give 9a. In Formula 8, Q can be a let" -,group. Q can be any cf the leaving groups discussed abovefor Group hl The compounds of formula 8 where Q = Cl- or Clare commercially available from EMS Dottikon, Lenzburg,Switzerland and Sugai Chemical Industries, Ltd. in hapan.

The coupling can be canned out in a variety of ways,depending on the identity of Q. When a free acid is used (Q= · , the coupling can be performed using carbodiv methods utilizing any of the common reagents of thisclass including dicyclohexylcarbodiimide or relateddialkylearbodiimides, EDC (salts of 1-(3- cim&amp;thylaminopropyli -3-ethylcarbodiimide) or relateu watersoluble reagents along with an organic amine base in polarc ..c solvents such as dioxane, DMF, NMP and acetonitrilein the presence of an K-hydroxyheterocvclic including 21-hydroxysuccinimide or 3-hydroxybenzotriazole. When f - aharogen or pseudohalogen, the coupling may be per* in moot common organic solvents such as THF, diethyl dioxane, methyl t-butyl ether or other ethers; acetone»cyclohexanone, methyl isobutylketone and other ketcu- :esters such as ethyl, methyl and isopropyl acetate;

8*910/66 /d/dV SUBSTITUTE SHEET (RULE 26) ΑΡ ΰ 0 13 5 8 ./*' 'f) -28- halogenated solvents such as halogenated methanes andethanes, chlorobenzene and other halogenated benzenes;nitriles such acetonitrile and propionitrile; lower alcoholssuch as ethanol, isopropanol, t-butanol and relatedalcohols, and polar organic solvents such as dimethylformamide, dimethylsulfoxide, N-methyl-2- pyrrollidone and related amide-containing solvents. A baseis frequently used and may be any of a number of inorganicbases such as metal hydroxides, bicarbonates and carbonatesor organic bases such as amines like triethylamine,diethylamine, diethyl isopropylamine, DABCO or related di-or trialkylamines, as well as amidine bases like DBU andDBN.

Acetate removal is accomplished in step 6b with aqueousor alcoholic solutions of inorganic bases such as metalhydroxides, carbonates and bicarbonates at ambienttemperatures up to 100 °C. If there is a protectedfunctionality on the carboxamide group bonded to theperhydroisoquinoline ring system, it is best removed at thispoint (during or after step 6b). The nature of this step is dependent on the exact identity of the protecting group. A preferred method for accomplishing the entire processshown in Scheme I is shown in Scheme II.

The Cbz-protected amino acid 15 was coupled with the amine22 to give the amide 16. The Cbz group was removed byhydrogenation to AP/P/ 9 9/01648 SUBSTITUTE SHEET (RULE 26) AP u0135 8

St'hvRM· II- Synthesis of Amick 21 com

Cta

CtjGH '-N-X J CNaq.HC! X,--·, oh .....— *<?r’V0rv*HXjuiiM*hnf .xb’h ί .. * '

Cnuniairoiisi HOx ί’Λ STEP 0

' . CtaG

STEP A S PS:hT- i , SPit Qv ,ρρ

CtriJN^jV' \ at 0 .0 ‘ .....! onus

HjN W \ p,"

EDO HOBfKjO

STEPB

50% ϋή. XtOK

STEPE

HV m h, e 5%Pd<

EtOK

STEPC XSPI> 0^, f< x. ' o >C«) ;: t-V Kt ‘ V ·

N 'V OH k Λ"

”1. J 19 τη; ίισι ;.ίϊ3 $(?/< Άϊ NOi

STEPF „SPh

Me 0 <

H SPi, N . • mPmc

N H °H <

K «·ΰ 2! give the amine 17. This was coupled with the chloroalcoholvis she epoxide using the in situ procedure to giveadduct. 18. Conventional deprotection with base and couplingof the free primary amine with the acid chloride 20 gaverise to amide 21. Details of this process are providedthaw in. Examples 1 A to F. The lettering A to F on SchemeII corresponds to Examples 1 A to F below.

The following Examples illustrate aspects ofinvent loro. These examples are for illustrative purposes andare not intended to .lootit the scope of the invention.

Abbreviations for the terms melting point, nucievrmagnetic resonance spectra, electron impact mass p - .field desorption mass spectra, fast atom bombardment massspectra, infrared spectra, ultraviolet spectra, e > calanalysis, high performance liquid chromatography, ano thinlayer chromatography are, respectively, m.p,, NMR, TIMS, MS-FD;, MS{FAB), IE, Uvo Analysis, HPLC, and TLC. toaddition, the absorption maxima listed for the IE stectraaxe tnose of interest, not all maxima observed, AP/P/ 99/01648 SUBSTITUTE SHEET (RULE 26) APV01358 -30-

In conjunction with the NMR spectra, the followingabbreviations are used: singlet (s' , doublet (d) , doubletof doublets (dd) , triplet (t) , quartet (q) , multiplet (τη) ,doublet of multiplets (dm), broad singlet (br.s), broaddoublet (br.d), broad triplet (br.t) , and broad multiplet(br.m). J indicates the coupling constant in Hertz (Hz).Unless otherwise noted, NMR data refer to the free base ofthe subject compound. NMR spectra were obtained on a General Electric QE-300300MHz instrument. Chemical shifts are expressed in δvalues in ppm. Mass spectra were obtained on a VG ZAB-3Spectrometer at the Scripps Research Institute, La Jolla, CA. Infra-red spectra were recorded on a Midac Corporationspectrometer. UV spectra were obtained on a Varian.Cary 3Einstrument. Thin layer chromatography was carried out usingsilica plates available from E. Merck. Melting points weremeasured on a Mettler FP62 instrument and are uncorrected.

Example 1

Procedures for the Synthesis of Amide of Formula 21 [3S-[2(2S*,3S*),3 alpha,4a beta,8a beta] ]-N-(1,l-dimethyl-2-hydroxyethy1) decahydro-2-[2-hydroxy-3-[(3-hydroxy-2-methylbenzoyl)amino]-4-(phenylthio)butyl] -3-isoquinolinecarboxamide

8*9 I 0 / 6 6/d/dV

H

,SPh CL A HO. A. Perhydroisoquinoline (26.4 g, 111 mmol) (commericallyavailable from NSC Technologies (Chicago, IL) or Procos SpA(Milan, Italy)) was suspended in water (200 mL) andconcentrated aqueous HC1 (200 mL). This mixture was heated SUBSTITUTE SHEET (RULE 26) ΑΡΰ01358 -31- 6ο reflux and stirred for 3 days, during which time it went,into solution. The solvents were removed under reducedpressure to give a light yellow solid. The solid wasslurried in 2-propanol (200 mL) and filtered. The filtratewas evaporated under reduced pressure to an oil. BoQAe QOOrnlf and water (100 mil were added and the pH of the solutionwas brought to 8.0 by the addition of 2 N aqueous KOH.

Bencyl chloroformate (15.8 mL, 111 mmol) was added dropwiseover 30 minutes and she pH was kept between 7 and 8 by theaddition of 2 N aqueous KOH. The mixture was stirrea atroom temperature for 16 hours. EtOAc (200 mL) was added andthe organic layer was washed with 1 N aqueous HCl ¢100 mL; ,and brine (100 mL) . The organic layer was dried (!· ; , filtered, and evaporated under reduced pressure tc an oil.The product was purified by silica gel chromatograph·/,eluting with 1;1 40-00 petroleum ether/StOAc followed by100·% EtOAc. The fractions containing product were collectedand evaporated under reduced pressure to give the : 'y....'urdIS (11.2 g, 32%) as 6 colorless oil: NMR (300 MHz, CDC1-) «5 ^.43-7..28 (m, 5 H) , 1,17 (br s, 2 H) , 4.76 £m, 1 H) , 3.79Cm, 1 K) , 3.33 (m, 1 Hu, 2.19 (m, 1 H) , 1.96 (in, 1. Ka 1..8S-1.15 inn 10 H) ., 5. 1-Hydroxybenzotriazole (4.2 g, 31.4 mmol) and EDO (6.0 g, 31.4 mmol) were added to a solution of acid 15 (3.3 g,20..2 mmol) in DMF (128 mL) at ambient temperature. fetenurture was heated at C for 10 minutes. 1,1 -Dimethyl-2- trimethylsilyloxyethylamine (5.1 g, 31.4 mmol, prej. from 1,1-dimethyl-2-hydroxyethylamine (Aldrich Chemical Col andhexamethyl di si la zane (Aldrich Chemical Co.)) by ?tr-- the mixture neat under reflux for several hours followevaporaticn of the volatile components was added ana thesolution was heated at 80° C for 17 hours. The yeiCavsolution was poured into EtOAc (250 mL) and 2 N aqueous ECl.¢250 mL). After stirring for 10 minutes EtOAc (750 mL) wasadded and the mixture was washed with H,0 (3 x 500 nin andb.rc.ne. (3. x 250 mL) . The combined aqueous layers wereer.uracted with EtOAc (1 x 250 mL) . The combined c, vlayers were dried (Na2S0.y and purified by flash

9 t 0 / 6 ♦» /uZdV SUBSTITUTE SHEET (RULE 25) AP V 0 13 5 8

-32- chromatography {50/50 EtOAc/hexanes) to give the compound 16as a colorless oil (7.9 g, 78%) : NMR (300 MHz, CD3OD) δ 7.36 (m, 5 H) , 5.20 (d, J = 8.1 Hz, 1 H) , 5.10 (m, 1 H) , 4.53 (m, 1 H), 3.78 (dd, J = 13.2, 4.4 Hz, 1 H), 3.60 (m, 2H), 3.48 (d, J = 10.7 Hz, 1 H), 2.15-1.25 (m, 12 H) , 1.31(s, 3 H) , 1.29 (s, 3 H) . C. A mixture of carbamate 16 (7.9 g, 20.4 mmol) and 5% palladium on carbon (Pd/C)(1.6 g) was hydrogenated at 50 psiH2 in absolute EtOH (110 mL) at ambient temperature for 18hours. The mixture was filtered through Celite andevaporated in vacuo to give amine 17 as a white, crystallinesolid: JH NMR (300 MHz, CD3OD) δ 3.63 (q, J = 7.0 Hz, 2 H) ,3.34 (m, 1 H), 3.27 (dd, J = 11.8, 3.3 Hz, 1 H), 2.91 (m, 1H), 2.02-1.15 (m, 12 H), 1.32 (s, 3 H), 1.31 (s, 3 H). D. Aqueous 10.2 N NaOH (2.4 mL, 24.5 mmol) was added to a warm (27 °C) suspension of chloroalcohol (obtained fromKaneka Industries in Japan)(10.4 g, 28.6 mmol) inisopropanol (IPA)(104 mL) with mechanical stirring. After 1hour 1 N aqueous HC1 in IPA (prepared by addition of 1 mL ofconcentrated aqueous HCl to 12 mL of IPA) approximately(ca.) 1 mL) was added to neutralize (pH = 7). Amine 17 (5.2g, 20.4 mmol) was added as a solution in IPA (50 mL) and thethin suspension was heated at 60° C for 10 hours. The IPAwas removed in vacuo. The residue was diluted with EtOAc(150 mL) and washed with H2O (2 x 50 mL) , saturated aqueousNaHCO3 (1 x 50 mL), and brine (1 x 50 mL). The combinedaqueous layers were extracted with EtOAc (1 x 25 mL). Thecombined organic layers were dried (Na2SO„) and purified byflash chromatography (75/25 EtOAc/hexanes, then EtOAc) togive the compound 18 as a white solid (8.98 g, 76%): ‘H NMR(300 MHz, CD3OD) δ 7.33 (m, 10 H) , 5.08 (AB, JM = 12.2 Hz,ΔυΑΒ = 12.1 Hz, 2 H) , 3.96, (m, 2 H) , 3.56 (q, J = 7.3 Hz, 2 H), 3.50, (m, 1 H), 3.20 (dd, J = 13.6, 9.2 Hz, 1 H), 3.03 (m, 1 H), 2.64 (m, 2 H), 2.20-1.20 (m, 14 H), 1.28 (s, 6 H).

8*910/66 /d/dV SUBSTITUTE SHEET (RULE 26) -33- Ξ. 50% aqueous NaOK (1.7 g, 1-8 mL, 33.6 mmol) was addedco a suspension of carbamate 18 (6.75 g. 11.6 mmol) in 1PA(33 mb) at ambient temperature. The mixture was he-.unaer reflux for 12 hours. After cooling to ambienttemperature, the mixture was diluted with methyl tether (ΜΤΒΞ) (600 mb) and washed with H20 (2 x 250 mid, and.bnae (1 x 125 mL) . The combined aqueous layers wereextracted with MTBE (1 r 150 mL). The combined organiclayers were dried (ha . and evaporated in vacuo to give a rnLxx ure of compound 19 and benzyl alcohol as an oiii- te so lia : T( NME (300 MHc , CD3OD) δ 7.34 (m, 10 H) , 4,11 (s, 2 HO , 3 , e i i rr;, 1 H) , 3.53 ; (m, 3 H) , 3 . 03--2.60 (m, 5 H)1 2.17 irn, 1 E; , 2.05 (m, j. E; , 1.87-1.05 (m, 12 H), 1. . r 0 ; x , .3 n; 1 iO is, 3 H) . F, Triethylamine (3.2 g, 4.3 mL, 31.2 mmol) was added to asolution of the mixture of amine 19 (4,7 g, 10.4 mmol tneoryfrom 18) and benzyl alcohol in EtOH (23 mL) at ambienttemperature, A solution of 3-acetoxy-2-methylbenzoychloride (20)(obtained according to procedures set forth inU.S Patent Application Serial No. 08/708,411, filedSeptember 5, 1996, which, is specifically incorporated byreference herein) {2.4 g, 11.5 mmol) in THF (4 mb) wasadded. After 2 hours aqueous NaOH (4.1 g, 2.8 no,, .52.2 mmol) was added and the mixture was heated under reflux for1 hour. After cooling to ambient temperature, the mixturewas neutralized to pH - " with 2 N aqueous HC1 (26 nT ,

This mixture was diluted with EtOAc (500 mb) and uu.- - withHoc (1 x 250 mb), saturated aqueous NaHCO3 (2 x 250 mis , H,0(1 x 25 0 mb) , ana brine (1 x 12 5 mL) . The organic 1 . wasdried iNa,3O.) and purified by flash chromatography (71/25EtOAc/hexanes) to give amide 21 as a white foam (IVAA-xll'A,1-11 a, 23%; , The lH NME. indicated the presence of 53 ot % . could, not be removed in vacuo. AP/P/9 9 *0 16 48 SUBSTITUTE SHEET (RULE 26) ΑΡ Ο 0 13 5 8 -34-

Analysis: *H NMR (300 MHz, CD3OD) δ 7.53 (d, J = 7.3 Hz, 2 H), 7.32 (t, J = 7.0 Hz, 2 H), 7.20 (t, J = 7.3 Hz, 1 H) , 7.06 (t, J = 8.1 Hz, 1 H), 6.92 (d, J = 8.1 Hz, 1 H), 6.83 (d, J = 8.1Hz, 1 H), 4.42 (m, 1 H), 4.08 (m, 1 H), 3.61 (dd, J = 13.6,4.0 Hz, 1 H) , 3.45 (AB, JM = 11.0 Hz, ΔυΑΒ = 18.0 Hz, 2 H) ,3.29 (dd, J = 13.6, 10.3 Hz, 1 H), 3.10 (m, 1 H), 2.66 (m, 2H) , 2.28 (s, 3 H), 2.22 (m, 2 H), 2.04 (m, 1 H), 1.86-1.20 (m, 11 H) , 1.19 (s, 3 H) 1.18 (s, 3 H) • 13C NMR (75.5 MHz, CD3OD) δ 175.7, 172.5 , 155.9, 138.8, 136 • 7, 129.8, 128.9, 126 .3 , 126.0, 122. 4, 118.4, 115.9, 70 . 3, 69.9, 68.2, 59.3, 58 .8, 54.9, 53. 0, 36.5, 34.2, 34.1 31. 1, 30.7, 26.4, 26.0, 23 .1, 23.0, 20. 8, 12.1.

Example 2 HIV Protease inhibition activity and anti HIV activity incell culture of compound 21

Tight binding kinetics analysis was used to determinethe magnitude of the ΚΛ values of compound 21. The K3= 5.6 +0.91nM.

Methods

Expression of HIV-1 protease HIV-1 protease gene was isolated from the viral strainIIIB (Ratner, L. et al., Nature, 316, 227-284 (1985)). Inorder to increase the stability of purified protease (Rose,J.R. et al., J. Biol. Chem., 268, 11939-11945 (1993)), theglutamine residue at position 7 (Q7) was mutated to serine(S) by replacing the 33 base pairs segment between the Ndeland BstEII sites of the protease gene sequence withsynthetic oligonucleotides encoding the Q7S mutation. Themodified gene sequence was inserted into the plasmid vectorpGZ (Menge, K.L. et la., Biochemistry, 34:15934-15942 (1995)under the control of phage T7 promoter. The resulting

8> 9 I 0 / 6 6 Zd/dV SUBSTITUTE SHEET (RULE 26) APi¢13 5 8 -35- cor:3"ruct, pGZ/HP-1 . was transformed into E. ccii strain BL21(DE3) purchased from Novagen, Inc.

Expression of HIV-1 PR: Cultures were grown imedia (1,.6% Trypticase Pepton, 1% Yeast extract, 0.1 % Nadat an initial pH 7.5} containing 200 pg/L ampicillir m 100L tormentor (Biolafitte, SA) at 37“C for 5 hours and theninduced by addition oil mM IPTG (Isopropyl-β-D- throgalactopyranoside}. The temperature of the cultureduring induction was raised to 42°C to increaseuccumulations of the recombinant HIV-1 protease as „ aubleinclusion bodies. After 2 hours at 42cC, cells werenarvssted by crossflow filtration using Pellicon 0.1 ;:ηVVPP000C5 cassette «10 (Millipore) and the cell paste wasstored frozen at -70‘hl

Purification of Recombinant HIV-1 Protease: Ail stepsunless otherwise indicated were carried out at 4f’C. Proteinconcentrations were determined using BioRad protein assaysolution with bovine serum albumin (BioRad, Richmond. CA) asa standard. Chromatographic steps and the purity of HIV PRwas analyzed by sodium dodecylsulfate polyacrylamide gelelectrophoresis (SDS-PAGE) . Final purity of HIV-P.R was> . 'Typical final yield from each 100 L culture was ~l20 mg .

Cell paste from 100L culture was resuspended in 300 mi,of lysis buffer (50 mM 7ris-Cl pH 8.0, 25 mM NaCl, 2C :το4 2--mercaptoethanol) and ncrofluidized in MicrofiuidicsCorporation fiuidizer at 22,000 psi. The crude cell lysatewas clarified by centrifugation at 14,000 rpm for 20minutes. HIV PR was found predominantly in the pellet, an theform cf inclusion la The inclusion bodies were subsequently washed - tie times in the lysis bufita;-cohtsxnxng in addition 0.1% Trition-XlOO and 1 M urea, andafter each washing ' . ure, the inclusion bodies tarot

8*910/66 Zd/dV SUBSTITUTE SHEET (RULE 26) ΑΡΟ 0 13 5 8 -36- pelleted by centrifugation at 5,000 rpm for 20 minutes.Purified inclusion bodies were solubilized in buffercontaining 50 mM Tris-Cl, pH 8.0, 25 mM NaCl, 20 mM 2-mercaptoethanol, and 8 M urea. Solution was clarified bycentrifugation at 14,000 rpm and applied at room temperatureto a 300 mL Fast Flow Q-Sepharose column (Pharmacia,Piscataway, NJ) equilibrated with the same buffer. Underthese conditions HIV PR did not bind to the column andessentially pure enzyme was found in the flow-throughfractions. To renature the protein, the fractions from FastFlow Q-Sepharose column were dialyzed against three changesof buffer containing 25 mM NaH2PO4 pH 7.0, 25 mM NaCl, 10 mMDTT and 10% glycerol. After refolding, small quantities ofprecipitated material were removed by centrifugation andresultant enzyme preparation were concentrated, dialyzedagainst 0.5 M NaCl, 50 mM MES pH 5.6, 10 mM DTT, frozen insmall aliquots at ~2 mg/mL and stored at -70°.

Tight-Binding Kinetics Assay and Analysis

Proteolytic activity of purified HIV-1 protease wasmeasured using a modified chromogenic assay developed byRichards at al. (Richards, A.D. et al. J. Biol. Chem., 256,773-7736 (1990)). The synthetic peptide His-Lys-Ala-Arg-Val-Leu-Phe(paraN02)-Glu-Ala-Nle-Ser-NH2 (American PeptideCompany) (Nle is norleucine) was used as a substrate. Theassay was carried out in 0.5 M NaCl, 50 mM MES pH 5.6, 5 mMDDT, and 2% DMSO at 37°C. Cleavage of the scissile bondbetween leucine and paranitro-phenylalanine (Phe para-N02)was assayed by spectrophotometric monitoring of the decreaseon absorbance at 305 nm. Initial velocity was determined asthe rate of decline of absorbance during the first 100seconds of the enzymatic reaction. Under these conditions,and using Q7S HIV-1 protease, the Michaelis constant (Km)for this substrate is 59 + 17 μΜ.

8*910/66 /d/dV SUBSTITUTE SHEET (RULE 26)

Ar -01358 -37 -

For determination of the inhibition of compound 21, asaturating concentration of substrate of 200 uM was used.Between 13 and 20 concentrations of inhibitors wereevaluated and the velocity of reaction was measured at eachconcentration as described above. The apparent Ki (Ki app) ,set forth above, was determined by computer assisted non-linear least square fitting of the data to the tight bindingequation of Morrison (Morrison, J.Ft, Biochem. B- t,

Acta, 185, 269-286 (1963;}.

Example____3

Antiviral activity of compound 21 against HIV-1 in cellculture

Cells and virus strains.:

The CEM-SS and MT-2 human T cell lines and C.3V- lstrains RF and IIIB were obtained from the AIDS Research andReference Program, Division of AIDS, KIAID, and NIH.

Ce ll_pr o t e ct wn__assaytt

The inhibitory effects of each agent on HIV-1 replication were measured by the MTT dye reduction method(Alley, M.C. et al. , Cancer Res. 48.: 589-601 (1988))-,Compounds were dissolved in DMSO at a concentration cl90 mg/ml then diluted 1:200 in culture medium (RPMIsupplemented with 10% fetal bovine serum). From eachdiluted stock, 100 μϊ was added to a 96-well plate andserial half-log dilutions were prepared. In separate tubes,MT-2 cells and CEM-SS cells were infected with HIV-l IIIB orKTC-1 RF at a multiplicity of infection (m.o.i.) of 3.01 and0 03, respectively. Following a 4-hour adsorption period,100 ul of infected or uninfected cells were added to thewells or the drug containing plate to give a finalconcentration of 1 x 10" cells/well. Six days (CEM£'Scells) or 7 days (KT-C: cells) later, MTT (5 mg/ml) was addedto test, plates and ount of formazan produced vto

8*910/66 /d/dV SUBSTITUTE SHEET (RULE 26) ΑΡ ϋ0 13 5 8 -38- quantified spectrophotometrically at 570 nm. Data wereexpressed as the percentage of formazan produced in drug-treated cells compared to formazan produced in wells ofuninfected, drug-free cells. The ED50 was calculated as theconcentration of drug that increased the percentage offormazan production in infected, drug-treated cells to 50%of that produced by uninfected, drug-free cells.

Cytotoxicity iTC50) was calculated as the concentration ofdrug that decreased the percentage of formazan produced inuninfected, drug-treated cells to 50% of that produced inuninfected, drug-free cells. The therapeutic index (TI) wascalculated by dividing the cytotoxicity (TC50) by theantiviral efficacy (ED50) .

Table 1

Antiviral Activity and Cytotoxicity Evaluations of Compound 21

in an Acute Infection of CEM-SS cells with HIV-1 RF

Compound ed50 (nM) ed95 (nM) tc50 (μΜ) Therapeuticindex * 21 34.2 154.1 96.6 2825 a z i dophymi dine(AZT) 52.3 543.1 >374.5 >7161 dideoxycytidine (ddC) 94.70 142.0 37.69 398 AP/P/ 9 9/01648 a Therapeutic index = Cytotoxicity (TC50) -i- Antiviralactivity (ED50) · SUBSTITUTE SHEET (RULE 26) ΑΡ ύ Ο 13 5 8

Table 2 ν'2ίΧΉ.1 AC ι. Σ Vi, υ V 3' in an Acute Inre ytotoxicity Evaluations r eu . i 21

5.n of MT-2 cells with HIV-1 ;IIB ) Compound !'c ' (nM) ed95 (nM) TC50 ίμΜ) T I............................... ND 92.6 «. J / '7 ND 109.4 j ddC i ND 176.3 " Therapeutic index .oxicity (TC^y v Antiviral activity (ED^q).

As noted above, the compounds of the present inventionare useful for inhibiting KIV protease, which is an emcymeassociated with viral component production and ay.*··. . An eniDodirnent of the present invention is a method of creatingHIV infection comprising administering to a host or patient,such as a primate, an effective amount of a compound offormula (9) or a pharmaceutically acceptable salt thereof.Another embodiment of the present invention is a me - oftreating .AIDS comprising administering to a host o ->ei *an effective amount of a compound of formula (9) or epharmaceutically atv .he salt thereof. A furtherembodiment of the present invention is a method oftmioiting KIV protease comprising administering to an HIVuhected cell or a -. ·. ,r patient, such as a prir,-inhected with KIV, -. :ective amount of a compou:formula )1) or a pharmaceutically acceptable salt · _ f

The term "effect tvs amount" means an amount of ocompound of formula (3) or its pharmaceutically .-. APZP/ 9 9/01648 SUBSTITUTE SHEET (RULE 26) AP001358

-40- salt that is effective to inhibit the HIV protease mediatedviral component production and assembly. The specific doseof compound administered according to this invention toobtain therapeutic or inhibitory effects will, of course, bedetermined by the particular circumstances surrounding thecase, including, for example, the compound administered, theroute of administration, the condition being treated andthe individual host or patient being treated. An exemplarydaily dose (administered in single or divided doses)contains a dosage level of from about 0.01 mg/kg to about 50mg/kg of body weight of a compound of this invention.Preferred daily doses generally are from about 0.05 mg/kg toabout 40 mg/kg and, more preferably, from about 1.0 mg/kg toabout 30 mg/kg.

The compounds of the invention may be administered by avariety of routes, including oral, rectal, transdermal,subcutaneous, intravenous, intramuscular and intranasalroutes. The compounds of the present invention arepreferably formulated prior to administration. Therefore,another embodiment of the present invention is apharmaceutical composition or formulation comprising aneffective amount of a compound of formula (9) or apharmaceutically acceptable salt thereof and apharmaceutically acceptable carrier, such as a diluent orexcipient therefor.

The active ingredient preferably comprises from 0.1% to99.9% by weight of the formulation. By "pharmaceuticallyacceptable" it is meant that the carrier, such as thediluent or excipient, is compatible with the otheringredients of the formulation and not deleterious to thehost or patient.

Pharmaceutical formulations may be prepared from thecompounds of the invention by known procedures using known AP/P/ 9 9/01648 SUBSTITUTE SHEET (RULE 26) ΑΡ Ο Ο 13 5 8 -41- ana readily available; ingredients. In making the compositions of the present invention, the active - its t rill usually be admixed with a carrier, or diluted by acarrier, or enclosed within a carrier, which may be in thefore of a. capsule, sachet, paper or other suitablecontainer. When the carrier serves as a diluent, it ray bea solid, semi-solid or liquid material which acts as aver.icie, excipient turn for the active ingredient

Thus, the compositions can be in the form of tablets, pills,powders, lozenges, sachets, cachets, elixirs, suspensions,emulsions, solutions, syrups, aerosols (as a solid o.c in aliquid medium), ointments (containing, for example, up co101 by weight of the active compound), soft and hard gelatincapsules, suppositories, sterile injectable solutisterile packaged powders and the like.

The following formulation examples are illustrativeonly and are not intended to limit the scope of theinvention. The term ”active ingredient" represents acompound of formula (9) or a pharmaceutically acceptablesal t thereof. AP/P/ 9 9/01648 SUBSTITUTE SHEET (RULE 26) ΑΡ Ο 0 13 5 8 -42-

Formulation 1

Hard gelatin capsules are prepared using the followingingredients :

Quantity(mg/capsule) 250 200 4 60 mg

Active ingredientStarch, driedMagnesium stearateTotal

Formulation 2 A tablet is prepared using the ingredients below:

Quantity (mg/tablet)

Active ingredient 250

Cellulose, microcrystalline 400

Silicon dioxide, fumed 10

Stearic acid 5.

Total 665 mg

The components are blended and compressed to form tablets each weighing 665 mg.

8*910/66 /d/dV SUBSTITUTE SHEET (RULE 26) APO 0 1358 -4 β- ίοι aerosol solution is prepared, containing the foil. coTiipoTtents :

Wj^iaht lowing

Active ingredient 0.25

Methanol 25.75 1 i o.ti r luorome’.i .. 74.00

ToIa 100.00

The active compound is mixed with ethanol and the mxxc!added to a portion of the propellant 22, cooled to - 3 Intransferred to a filling device. The required amountfed to a stainless steel container and diluted with tvremainder of the propellant. The valve units are thento the container. £unatdni£ipn_,£

Tablets, each containing 60 mg of active ingredient'made as follows:

Quantity imo/tablet)

Active ingredient 60

Starch 45

Miorcnryscalline cellulose 35

Polyvinylpyrrolidone 3 C and i3 then fitted are AP/P/Q9 '01648 \ clip 10% solution in water; 4 Sodium i carboxymethyl starch 4.5 Magnet rum stearate 0.5 1' Η X C 1 Total 150 SUBSTITUTE SHEET (RULE 26) ΑΡ Ο Ο 13 5 8 -44-

The active ingredient, starch and cellulose are passed througha No. 45 mesh U.S. sieve and mixed thoroughly. The aqueoussolution containing polyvinylpyrrolidone is mixed with theresultant powder, and the mixture then is passed through a No.14 mesh U.S. sieve. The granules so produced are dried at 50°Cand passed through a No. 18 mesh U.S. sieve. The sodiumcarboxymethyl starch, magnesium stearate and talc, previouslypassed through a No. 60 mesh U.S. sieve, are then added to thegranules which, after mixing, are compressed on a tabletmachine to yield tablets each weighing 150 mg.

Formulation.,, 5

Capsules, each containing 80 mg of active ingredient, aremade as follows:

Quantity (mo/capsule)

Active ingredient 80 mg Starch 59 mg Microcrystalline cellulose 59 mg Magnesium stearate mg Total 200 mg AP/P/ 9 9 / 0 1 6 48

The active ingredient, cellulose, starch and magnesiumstearate are blended, passed through a No. 45 mesh U.S. sieve,and filled into hard gelatin capsules in 200 mg quantities.Formulation 6

Suppositories, each containing 225 mg of activeingredient, are made as follows:

Active ingredient 225 mg

Saturated fatty acid glycerides 2.000 mg

Total 2,225 mg

The active ingredient is passed through a No. 60 mesh U.S. sieve and suspended in the saturated fatty acid glycerides previously melted using the minimum heat necessary. The SUBSTITUTE SHEET (RULE 26)

Ar Ο Ο 1 3 5 8 mixture is then poured into a suppository mold of nominal 2 g

capacity and allowed to cool , formulation i7 Suspensions, each , containing 50 mg of active ingc·.·..... per 5 mi dose, are made as follows: Active ingredient 50 mg Sodium carboxymethyl ce Iiulose 50 mg Syrup 1.25 ml Benzene acid solution 0.10 ml Flavor q . v. Col or q.v. Purified water to total 5 ml The active ingredient i s passed through a No, 4 5 mesh IAS sieve and mixed with the sodium carboxymethylcellulosesyrup to form a smooth paste. The benzoic acid solution,flavor and color are diluted with a portion of the water andadded, with stirring. Sufficient water is then added toproduce the required volume.

EaXMlihinon „g.

An intravenous formulation is prepared as follows:

Active ingredient 10c mg

Isotonic saline 1,000 nil

The solution of the above ingredients generally is administered intravenously to a subject at a rate of 1 per minute .. AP/P/ 9 9/01648 SUBSTITUTE SHEET (RULE 26) ΑΡ Ο 0 13 5 8 -46-

Pormulation 9 A tablet is prepared using the ingredients below:

Quantity (mo/tablet) Active ingredient 2 92 mg calcium silicate 14 6 mg crospovidone 14 6 mg Magnesium stearate _5. mg Total 589 mg

AP/P/ 9 9 / 0 1 6 48 SUBSTITUTE SHEET (RULE 26)

Claims (7)

1. AP.O 0 13 5 8 -4 7- j. , A compound of the formula (9(:

   9 wherein: R and R' are independently selected from H, a substitutedor unsubstituted alkyl-OR, group, a cyolcalkyl osubstituted with a (Cl -Cy alkyl group or a (C,_-Ch alkyl-OHcroup, a heterocycle croup substituted with a (b-ly alkylcroup or a iC-C5) alkyl -OK group, an aikyi-RRyRj group, or analkyl - S ( X ( (Y) R, group , wherein ; ik is H, a substituted or unsubsituted alkyl group, or an acyl group; R, and F.i are each independently selected iron y,substituted or unsubstituted alkyl, cyoloalkyi,heterocycle, and aryl groups, and acyl and suifonylcroups; R, is H, a substituted or unsubstituted alkyl, cyoloalkyi,heterocycle, or aryl group; and X and Y are each independently selected from --1 and nothing, or a pharmaceutically acceptable salt or solvate thereof.
2. 2. A compound according to claim 1, wherein X, is H, ora pharmaceutically acceptable salt or solvate thereof. AP/P/ 98/01 648 ΑΡ Ο 013 5 8 -50-
3. 12. Use of a compound according to claim 1, or a pharmaceutically acceptable prodrug, salt or solvate thereof toinhibit HIV protease.
4. 13. Use of a compound according to claim 7, or a pharmaceutically acceptable prodrug, salt or solvate thereof toinhibit HIV protease.
5. 14 . A compound according to claim 1, which has a purity of more than 90% . 15 . A compound according to claim 1, which has a purity- of at least 95%. 16 . A compound according to claim 1, which a purity of at least 97%.
6. 17. A compound according to claim 1, which has a purity of at least 99%. 18. A compound according to claim 7, which than 90%. -H5.S S pOOL lV O2Z more 19. A compound according to claim 7, which least 95%. has a purity of ac 20. A compound according to claim 7, which least 97%. has a purity of at 21. A compound according to claim 7, which least 99%. has a purity of at 22. A pharmaceutical composition according the compound has a purity of more than 90% to claim 10, wherein 23. A pharmaceutical composition according the compound has a purity of at least 95% to claim 10, whe rein 24. A pharmaceutical composition according the compound has a purity of at least 97% to claim 10, whe rein 25. A pharmaceutical composition according the compound has a purity of at least 99% to claim 10, whe rein wherein 8 V 9 I 0 I 6 6 IdldV
7. 26. A pharmaceutical composition according to claim 11,the compound has a purity of more than 90%